This proposal represents a shift in direction from basic membrane domain studies in model and biological membranes in the previous grant to a commanding health-related problem, HIV infection, with strong connections to membrane lateral heterogeneity. Our overall goal is to apply cutting edge imaging technologies for the living cell to elucidate those aspects of virus infectivity that depend crucially on dynamic domains in the plasma membrane. In Specific Aim I, using molecular biology and lipid manipulation techniques in combination with live cell imaging, we will ask what molecular factors promote formation and stabilization of microdomains of influenza hemeagglutinin, HA, expressed in fibroblasts. This combined approach will provide live cell nanoassays for molecular interactions important in domain assembly and stability. Aim II will focus on the C-type lectin receptor DC-SIGN (CD209) which is expressed on the surface of Dendritic Cells (DC) where it functions as an antigen capture receptor and cell adhesion molecule. Various microbes, including HIV-1, bind to DC-SIGN to gain entry into DC. Previous studies using fixed cells demonstrated that DC-SIGN forms discrete antigen-capture clusters of 100-200 nm diameter on immature DC in the absence of any receptor ligation. We will study DC-SIGN clusters on living cells where detailed analysis of their dynamic properties--when loaded with different cargos--can be pursued. We will test the hypothesis that a key initial step in viral infection involves DC- SIGN clusters that, when loaded with pathogen cargo, are triggered to undergo directed lateral transport to sites of further processing by the host cell. Aim III will focus on microdomains that contain multiple components including the HIV-1 Gag polyprotein and a class of transmembrane proteins, the tetraspanins. We will test the hypothesis that the initial stages of virus assembly require Gag multimers to localize to tetraspanin-enriched microdomains and, once there, form stable clusters that will become sites of viral budding. PUBLIC HEALTH RELEVANCE The health-related significance of this proposal is based on the fact that effective, long-lasting strategies to thwart productive HIV-1 infection have remained elusive. The overwhelming majority of vaccine development has focused on the traditional methods of modifying the virus to elicit an immune response but this has been problematic because HIV-1 maintains one of the highest mutation rates amongst all viruses. As a result, increasing efforts have been focused on understanding how the virus interacts with its host cell with the ultimate goal of therapeutically disrupting these interactions. Cellular membrane microdomains are critical to the life of HIV-1 in cells in that the virus relies on their specific spatial arrangements for both entry into and exit out of target cells. Detailed information obtained in this grant on such membrane domains in the host cell will provide a better understanding of certain steps in HIV infection, thereby leading to new therapeutic targets.